The invention concerns a hedgehog protein conjugate with increased activity, a process for its production and its therapeutic use.
Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Pemmon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In the naturally occurring protein the N-terminal domain is modified with cholesterol at its C-terminus after cleavage of the C-terminal domain (J. A. Porter et al., Science 274 (1996) 255-259). In higher life-forms the hh family is composed of at least three members namely sonic, indian and desert hh (shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamum et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469).
Hynes et al. compare the activity of hh in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh produced from E. coli and find a four-fold higher activity of hh from the supernatants of the kidney cell line. The reason for this increased activity of hh has been discussed to be a potential additional accessory factor which is only expressed in eukaryotic cells, a post-translational modification, a different N-terminus since the hh isolated from E. coli contains 50% of a hh form which carries two additional N-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, or a higher state of aggregation (e.g. by binding to nickel agarose beads).
Nakamura et al. compare the activity of shh in the supernatant of transformed chicken embryo fibroblasts with an shh fusion protein isolated from E. coli which still has an N-terminal polyhistidine part. The shh in the supernatant of the fibroblasts has a seven-fold higher activity than the purified E. coli protein with regard to stimulation of alkaline phosphatase (AP) in C3H10T xc2xd cells. The increased activity has been postulated to be due to synergism of hh with molecules such as bone morphogenetic proteins (BMPS) which are only present in the supernatant of eukaryotic cells and in combination with hh cause the stronger induction of AP.
Kinto et al., FEBS Letters, 404 (1997) 319-323 describe that fibroblasts which secrete hh induce ectopic bone formation in an i.m. implantation on collagen. However, such an activity is not known for an isolated hh protein.
This invention provides a hedgehog conjugate, comprising from 1 to 4 moieties covalently bound to a hedgehog protein; wherein each moiety is: a) a polypeptide composed of 10 to 30 hydrophobic amino acids and/or of amino acids which form transmembrane helices and are positively charged, b) an aliphatic, saturated or unsaturated hydrocarbon residue with a chain length of 8 to 24 C atoms and with a hydrophobic action, or c) a hydrophobic thio group.
The object of the invention is to provide hh protein (polypeptide) conjugates which have a considerably improved activity compared to the known forms. The conjugates of this invention have the same utility as the corresponding unconjugated hedgehog protein.